Butanol is an important industrial chemical with a wide range of applications. It can be used as a motor fuel particularly in combination with gasoline to which it can be added in all proportions. Isobutanol can also be used a precursor to Methyl Tertiary Butyl Ether (MTBE). Currently the world production of n-Butanol is 3.5 million tons/yr (7.7 billion lb/yr). Furthermore, conversion of alcohols to long chain linear hydrocarbons that would be suitable for jet fuel use are being developed and demonstrated, which could further increase the demand for n-Butanol (The Naval Air Warfare Center—Weapons Division, (2012) Cobalt and Abermarle). For many centuries, simple sugars have been fermented into butanol with the help of Saccharomyces cerevisae. Fermentation of carbohydrates to acetone, butanol and ethanol (ABE) is well known and was commercially practiced worldwide from around 1915 to 1955 (Beesch, S. C. (1953) A Microbiological process Report—Applied Microbiology, 1, 85-95). With the advent of petrochemical processes and low cost petrochemical feedstocks the carbohydrate based processes became unattractive and were discontinued.
Further development and modernization of the ABE process was undertaken by several organizations. In the mid-1980s the Corn Products Corporation developed asporogenic strains and a multi-staged fermentation process that considerably improved the process economics (Marlatt, J. A. and R. Datta, (1986) Acetone-Butanol Fermentation Process, Biotechnology Progress (1986) 2, 1.23-28). Currently, two companies, Gevo and Butamax are engaged in conversion of several ethanol plants using recombinant microorganisms to produce iso-butanol for new chemical uses. See U.S. Pat. No. 8,017,375 and U.S. Pat. No. 7,851,188. In all of these developments the primary feedstock is carbohydrate, primarily starch from corn.
The limitations for carbohydrate feedstocks are well known and some are fundamental. Starch and sugars from agricultural crops run into competing issues of food vs. energy/chemical production as well as the cost of the feedstocks and their availability. For lignocellulosic feedstocks such as woody biomass, grasses etc. the cost and yield from pretreatment and hydrolysis processes are very limiting. For example, typical woody biomass contains 50% cellulose while the remainder consists of hemicelluloses, lignin and other fractions. The chemical energy content of the fermentable fractions is often less than 50% of that of the feedstock, putting fundamental limitations on product yield.
Attempts have been made to improve the alcohol yield of bacterium that ferment a variety of sugars to acetate and butyrate. The art has sought to employ recombinant techniques to transform bacterium such as C. acetobutylicum (Green et al. (1996) Genetic manipulation of acid formation pathways, Green et al. Microbiology), 142, 2079-2086) and C. tyrobutyricum (X. Liu et al. (2006) Construction and Characterization of ack Deleted Mutant of Clostridium tyrobutyricum, Biotechnology Pref., 22, 1265-1275). However, such techniques have only resulted in transformation occurring at low frequencies.
Several microorganisms are able to use one-carbon compounds as carbon source and some even as an energy source. Synthesis gas is a common substrate for supplying the one carbon compounds such as CO and CO2 and as well as hydrogen. Synthesis gas can be produced by gasification of the whole biomass source without the need to unlock certain fractions. Synthesis gas can also be produced from other feedstocks via gasification of: (i) coal, (ii) municipal waste (iii) plastic waste, (iv) petcoke and (v) liquid residues from refineries or from the paper industry (black liquor). Synthesis gas can also be produced from natural gas via steam reforming or autothermal reforming (partial oxidation).
When the syngas source is biomass, gasification technology converts all the components of the feedstock primarily to a mixture of CO, H2, CO2 and some residual CH4, typically with 75 to 80% cold gas efficiency i.e. 75 to 80% of the chemical energy of the feedstock is available for further chemical or biological conversion to target products. The rest of the energy is available as heat that can be used to generate steam to provide some or all of the process energy required. Furthermore, a wide range of feedstocks, both renewable such as woody biomass, agricultural residues, municipal wastes etc. or non-renewable such as natural gas, can be gasified to produce these primary components. Natural gas can be economically reformed to syngas with a wide variety of technologies using steam, oxygen, air or combinations thereof. This syngas has very good cold gas efficiency of approximately 85% to produce CO, H2 and CO2 with a wide range of target compositions.
Hence, syngas is a very economical feedstock that can be derived from a wide range of raw materials both renewable and non-renewable. Thus conversion of syngas to butanol with high yield and concentrations would lead to economical production of this important chemical.
The ability of anaerobic bacteria to produce n-butanol from the primary syngas components CO and H2/CO2 was discovered and reported in 1990/1991 by a team from the Michigan Biotechnology Institute, (A. Grethlein et al. (1991) Evidence of n-Butanol Production from Carbon Monoxide, Journal of Fermentation and Bioengineering, 72, 1, 58-60); (Grethlien et al. (1990) Continuous Production of Mixed Alcohols and Acids from Carbon Monoxide, Journal of Fermentation and Bioengineering, 24-25(1):875-885). Later, other organizations such as University of Oklahoma and Oklahoma State University also isolated new organisms namely Clostridium Carboxydivorans that also showed such conversion and n-butanol production (J. S. Liou et al. (2005) Clostridium Carboxidivorans sp. nov. a solvent producing clostridium Internation Journal of Systematic and Evolutionary Microbiology 55(5):2085-2091). Subsequent fermentation development with these and other organisms in single culture fermentations have not been very successful—the n-butanol concentrations were achieved in the range of approximately 3 g/liter and the yield ranged from 20 to 45% of theoretical (% electrons to product) (see previous three references and Guilaume Bruant et al. (2010) Genomic Analysis of Carbon Monoxide Utilization and Butanol Production by Clostridium carboxidivorans, PLoS One, 5(9)). For a commercially successful process, the n-butanol concentration should be in the range of 8-10 g/liter and the yield should be in the 80% range, otherwise processing and separations costs become unattractive.
To overcome these barriers multi-stage fermentations with two or more organisms such as Butyribacterium methylotrophicum and Clostridium acetobutylicum have been proposed (Worden et al. (1991) Production of butanol and ethanol from synthesis gas via fermentation, Fuel, 70, 6154-619). The former would produce butyric acid and butanol at low concentrations from syngas and the latter would uptake these while converting carbohydrates to produce more butanol. Since C. acetobutylicum strains are able to produce 15 g/liter butanol the separations process would be viable. Such a combination could provide some increases in yield and product recovery, but it would be very cumbersome requiring two different types of feedstocks, syngas and carbohydrates as well as separate bioreactors one for gas conversion and another for carbohydrate conversion. Furthermore, in this scheme the carbohydrate feeding the Clostridium acetobutylicum is the primary feedstock and not the more economical syngas fed to the Butyribacterium methylotrophicum and all the limitations of carbohydrate feedstocks described above will be prevalent.
A more efficient conversion of syngas takes place when converting it to ethanol and acetate. The biochemical pathway of such synthesis gas conversion is described by the Wood-Ljungdahl Pathway. Fermentation of syngas to ethanol and acetate offers several advantages such as high specificity of the biocatalysts, lower energy costs (because of low pressure and low temperature bioconversion conditions), greater resistance to biocatalyst poisoning and nearly no constraint for a preset H2 to CO ratio (M. Bredwell et al. (1999) Reactor design issues for synthesis-gas fermentations, Biotechnology Progress 15, 834-844); (Klasson et al. (1992), Biological conversion of synthesis gas into fuels”, International Journal of Hydrogen Energy 17, p. 281). Acetogens are a group of anaerobic bacteria able to convert syngas components, like CO, CO2 and H2 to acetate and ethanol the reductive acetyl-CoA or the Wood-Ljungdahl pathway.
Several anaerobic bacteria have been isolated that have the ability to ferment syngas to ethanol, acetic acid and other useful end products. Clostridium ljungdahlii and Clostridium autoethanogenum, were two of the first known organisms to convert CO, CO2 and H2 to ethanol and acetic acid. Commonly known as homoacetogens, these microorganisms have the ability to reduce CO2 to acetate in order to produce required energy and to produce cell mass. The overall stoichiometry for the synthesis of ethanol using three different combinations of syngas components is as follows (J. Vega et al. (1989) The Biological Production of Ethanol from Synthesis Gas, Applied Biochemistry and Biotechnology, 20-1, p. 781):6CO+3H2O→CH3CH2OH+4CO2 2CO2+6H2→CH3CH2OH+3H2O6CO+6H2→2CH3CH2OH+2CO2 
The primary product produced by the fermentation of CO and/or H2 and CO2 by homoacetogens is ethanol principally according to the first two of the previously given reactions. Homoacetogens may also produce acetate. Acetate production occurs via the following reactions:4CO+2H2O→CH3COOH+2CO2 4H2+2CO2→CH3COOH+2H2O
Clostridium ljungdahlii, one of the first autotrophic microorganisms known to ferment synthesis gas to ethanol was isolated in 1987, as an homoacetogen it favors the production of acetate during its active growth phase (acetogenesis)) while ethanol is produced primarily as a non-growth-related product (solventogenesis) (K. Klasson et al. (1992) Biological conversion of synthesis gas into fuels, International Journal of Hydrogen Energy 17, p. 281).
Clostridium autoethanogenum is a strictly anaerobic, gram-positive, spore-forming, rod-like, motile bacterium which metabolizes CO to form ethanol, acetate and CO2 as end products, beside it ability to use CO2 and H2, pyruvate, xylose, arabinose, fructose, rhamnose and L-glutamate as substrates (J. Abrini, H. Naveau, E. Nyns), “Clostridium autoethanogenum, Sp-Nov, an Anaerobic Bacterium That Produces Ethanol from Carbon-Monoxide”, Archives of Microbiology, 161(4), p. 345, 1994).
Anaerobic acetogenic microorganisms offer a viable route to convert waste gases, such as syngas, to useful products, such as ethanol, via a fermentation process. Such bacteria catalyze the conversion of H2 and CO2 and/or CO to acids and/or alcohols with higher specificity, higher yields and lower energy costs than can be attained by traditional production processes. While many of the anaerobic microorganisms utilized in the fermentation of ethanol also produce butanol as a secondary product, to date, no single anaerobic microorganism has been described that can utilize the syngas fermentation process to produce high yields of butanol.
Therefore a need in the art remains for methods using microorganisms in the production of butanol using syngas as the primary fermentation substrate.